Methods of identifying anti-viral agents

ABSTRACT

The invention provides methods to identify specific inhibitors of HPV E7 binding to CDK2 complex and methods to identify specific inhibitors of E7-induced CDK2 kinase activity. Specific inhibitors identified by the methods, compositions comprising the specific inhibitors, and methods of treatment using the compounds are also provided.

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/382,616 filed Aug. 25, 1999, which is pending.

FIELD OF THE INVENTION

[0002] The invention provides methods to identify anti-viral agents, andmore specifically, inhibitors of human papillomavirus E7 protein-inducedincrease in CDK2 kinase activity.

BACKGROUND

[0003] The human papillomaviruses (HPVs) are a family of more than 80small (approximately 8 kb) DNA viruses that infect stratified squamousepithelia causing warts. Certain high-risk HPV strains, including HPV16, HPV18, and HPV31, have been implicated as the most importantetiological agents in cervical cancer [zur Hausen, Biochim. Biophys.Acta 1288:F55-78 (1996)], which is consistent with the observation thatE6 and E7 genes from the high risk HPVs are potent oncogenes. Oncogenicpotential of E6 and E7 may arise from binding properties to host cellproteins. For example, E6 binds to the tumor-suppressor protein p53leading to ubiquitin-dependent degradation of the protein [Scheffner, etal., Cell 63:1129-36 (1990)], and E7 binds and promotes degradation ofthe tumor-suppressor retinoblastoma protein (pRb) [Dyson, et al.,Science 243:934-7 (1989); Jones, et al., Genes & Dev 11:2101-11 (1997)].While E6 and E7 have other activities, their roles in the viral lifecycle are not fully elucidated.

[0004] The HPV life cycle is regulated in a differentiation-dependentmanner within stratified-squamous epithelia [Jones, et al., Genes & Dev11:2101-11 (1997)]. The virus is maintained as an episome in the basalcell layer, which is the replicating cell population in stratifiedepithelia. With differentiation of the host cells into keratinocytes,the virus undergoes a burst of DNA replication. Followingdifferentiation, keratinocytes exit the cell cycle and die during thenormal course of epithelial stratification. These events are normallyirreversible, but HPV E7 activity is sufficient to promote anunscheduled round of DNA synthesis in differentiated keratinocytes[Cheng, et al., Genes & Dev. 9:2335-49 (1995)] The newly synthesizedviral DNA is packaged in the upper viable layers of the epithelia, andsloughed into the environment in the dead, differentiated cells. Theunscheduled DNA synthesis in differentiated cells is central to the HPVviral life cycle, and the E7 gene product has been implicated as a keyviral protein in this event. Consistent with these observations, recentgenetic analysis has shown that E7 is required for vegetativereplication (amplification) in HPV infected keratinocytes. The E7 geneproduct is a 98 amino acid protein that binds a number of regulatoryproteins, including pRb and proteins in the cyclin-dependent kinaseinhibitory protein (KIP) family, the function of which is critical forentry into S-phase entry of the cell cycle [Morgan, Ann. Rev. Cell Dev.Biol. 13:261-291 (1997)].

[0005] How E7 promotes progression into S phase has been the subject ofintense research because of the importance of this event to the virallife cycle and HPV-related cancer. E7 can overcome negative cellulargrowth signals including, for example, those mediated by TGF-β[Pietenpol, et al., Cell 61:777-85 (1990)], loss of substrate adherence[Ruesch, et al., Virol. 250:19-29 (1998)], and serum deprivation [Pei ,et al., Carcinogenesis 19:1481-6 (1998)]. This activity correlates, inpart, with the ability of E7 to transform cells and bind pRb familymembers [Galloway, et al., Semin. Cancer Biol. 7:309-15 (1996)]. E7binds other proteins, including, for example transcription factors suchas TATA-binding proteins [Massimi, et al., Oncogene 12:2325-30 (1996)],and c-jun and c-fos family members [Antinore, et al., EMBO. J.,15:1950-60 (1996)].

[0006] Despite these binding activities, it is unclear which knownfunction(s) of E7, if any, are key for the viral life cycle. Mostnotably, E7 binds pRb family members [Dyson, et al., Science 243:934-7(1989); Ciccolini, et al., Oncogene 9:2633-8 (1994); Wu, et al., J.Virol. 67:2402-7 (1993)], p21 [Funk, et al., Genes & Dev 11 :2090-100(1997); Jones, et al., Genes & Dev 11:2101-11 (1997)], and p27 [Zerfass,et al., Oncogene 13:2323-30 (1996)], proteins that participate in thecyclin-dependent kinase phosphorylation pathway regulating cell cycleprogression. The cyclin-dependent kinases regulate cell cycleprogression by a variety of means [Morgan, Ann. Rev. Cell Dev. Biol.13:261-291 (1997)], including inhibiting the ability of pRb to sequesterE2F [Mulligan, et al., Trends Genet 14:223-9 (1998)], a protein thatupregulates a variety of genes required for entry into S phase. E7binding to p21 and p27, both of which inhibit CDK phosphorylation,results in a net increase in CDK2 activity. These inhibitor proteinshave been implicated as key regulators of cell cycle progression thatact, at least in part, via a common cyclin-dependent kinase inhibitorydomain found in the amino terminus of these proteins [Polyak, et al.,Cell 78:59-66 (1994); Chen, et al., Mol. Cell. Biol. 16:4673-82 (1996)].E7 from viruses with low oncogenic potential lacks these bindingactivities, suggesting that interaction with one or more cellularproteins is important for neoplastic progression. Whether any of theseproperties are essential in the viral life cycle is unclear [Davies, etal., J. Virol., 67:2521-8 (1993); Funk, et al., Genes & Dev 11:2090-100(1997)].

[0007] Thus there exists a need in the art to more fully determinemechanisms by which E7 is able promote viral replication and to developmethods to identify inhibitors of E7 specific activity. Inhibition of E7can result in potent anti-viral activity and therefore, methods toidentify inhibitors of E7-dependent activity are desirable.

SUMMARY OF THE INVENTION

[0008] The present invention provides methods for identifying anti-viralagents. In a preferred embodiment, methods of the invention identifyagents that reduce or inhibit proliferation of human papillomaviruses.

[0009] The invention provides methods for identifying an inhibitor ofE7-induced CDK2 kinase activity comprising the steps of: a) measuringCDK2 kinase activity on a CDK2 substrate in the presence of humanpapillomavirus (HPV) E7, or a fragment thereof, and in the presence andabsence of a test compound, and b) identifying the test compound as aninhibitor of E7-induced CDK2 kinase activity when decreasedphosphorylation of the CDK2 substrate is detected in the presence of thetest compound compared to phosphorylation of the CDK2 substrate detectedin the absence of the test compound. The CDK2 kinase activity of theinvention is that which is detected from a CDK2/cyclin complex asdescribed and exemplified herein. In one aspect, the methods of theinvention include use of an E7 fragment that activates CDK2, wherein theE7 fragment consists of a continuous amino terminal fragment of E7beginning at amino acid residue 1 and terminating at a carboxy terminalresidue selected from the group consisting of amino acid residues 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, and 97 asset out in SEQ ID NO. 1. In a preferred embodiment, methods of theinvention comprising use of an E7 fragment selected from the groupconsisting of amino acid residues 1 to 27, amino acid residues 1 to 38,amino acid residues 1 to 48, amino acid residues 1 to 69, amino acidresidues 1 to 87 as set out in SEQ ID NO: 1. Methods of the inventionpreferably include a CDK2 substrate selected from the group consistingof histone H1, HPV protein E1, and HPV protein E2. Methods of theinvention include those in which E7 binding to a CDK2 kinase complex iseffected through the cyclin component of the CDK2 complex.

[0010] The invention also provide methods for identifying an inhibitorof E7-induced CDK2 kinase activity comprising the steps of: a) measuringCDK2 kinase phosphorylation of a substrate; b) measuring increased CDK2kinase phosphorylation of a substrate in the presence of humanpapillomavirus (HPV) E7, or a fragment thereof, to determine E7-inducedCDK2 kinase activity; c) measuring CDK2 kinase phosphorylation of asubstrate in the presence of HPV E7, or a fragment thereof, and in thepresence of a test inhibitor compound; and d) identifying the testcompound as an inhibitor of E7-induced CDK2 kinase activity when theincreased phosphorylation measured in step (b) is reduced in step (c)the presence of the test compound. In one aspect, the methods of theinvention include use of an E7 fragment that activates CDK2 wherein theE7 fragment consists of a continuous amino terminal fragment of E7beginning at amino acid residue I and terminating at a carboxy terminalresidue selected from the group consisting of amino acid residues 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68 ,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, and 97 asset out in SEQ ID NO. 1. In a preferred embodiment, methods of theinvention comprise use of an E7 fragment selected from the groupconsisting of amino acid residues 1 to 27, amino acid residues 1 to 38,amino residues 1 to 48, amino acid residues 1 to 69, and amino acidresidues 1 to 87 as set out in SEQ ID NO: 1. Methods of the inventionpreferably include a CDK2 substrate selected from the group consistingof histone H1, HPV protein E1, and HPV protein E2..

[0011] The invention also provides methods for identifying an anti-viralagent comprising the steps of: a) identifying an inhibitor of E7-inducedincrease in CDK2 kinase activity; b) measuring viral proliferation inthe presence and absence of the inhibitor identified in (a); and c)identifying the inhibitor as an antiviral agent when decreased viralproliferation is detected in the presence of the inhibitor compared toviral proliferation in the absence of the inhibitor.

[0012] The invention further provides methods for reducing humanpapillomavirus (HPV) E7-induced CDK2 kinase activity comprising the stepof contacting an HPV infected cell with an inhibitor of E7-induced CDK2phosphorylation. In another embodiment, the invention provides methodsfor reducing human papillomavirus (HPV) E7-induced CDK2 kinase activitycomprising the step of contacting an HPV infected cell with an inhibitorof E7 binding to CDK2 kinase complex.

[0013] The invention also provides methods for ameliorating humanpapillomavirus (HPV) proliferation comprising the step of administeringto an individual in need thereof an effective amount of an inhibitor ofHPV E7-induced CDK2 kinase activity. In another aspect, the inventionprovide methods for ameliorating human papillomavirus (HPV)proliferation comprising the step of administering to an individual inneed thereof an effective amount of an inhibitor of HPV E7 binding toCDK2 kinase complex.

DETAILED DESCRIPTION OF THE INVENTION

[0014] Methods of the invention include use of viral proteins thatinduce CDK2 kinase activity. Preferably, the viral proteins used inmethods of the invention are HPV E7 (as exemplified herein) or otherviral proteins which are at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99% homologous to E7 Cr1 and/or CR2 regions. Homology can bedetermined, for example, using basic BLAST search analysis with defaultparameters. Exemplary viral proteins with homology to the CR1 and/or CR2regions of E7 include adenovirus E1A and SV-40 T antigen.

[0015] The invention provides methods for identifying an inhibitor ofE7-induced CDK2 kinase activity comprising the steps of: a) measuringCDK2 kinase activity on a CDK2 substrate in the presence of humanpapillomavirus (HPV) E7, or an E7 fragment thereof, and in the presenceand absence of a test compound, and b) identifying the test compound asan inhibitor of E7-induced CDK2 kinase activity when decreasedphosphorylation of the CDK2 substrate is detected in the presence of thetest compound compared to phosphorylation of the CDK2 substrate detectedin the absence of the test compound. E7-induced CDK2 kinase activity isthe increased phosphorylation of a CDK2 substrate observed when the CDK2complex is contacted with E7 in the absence of a CDK2 kinase inhibitor(e.g., p21 and/or p27), compared to CDK2 substrate phosphorylationobserved in the absence of E7 and a CDK2 kinase inhibitor. Thus,optionally CDK2 kinase activity in the absence of E7 may be determinedas a control. In one aspect, the methods of the invention include use ofan E7 (including other viral E7 homologs or orthologs that induce CDK2activity) fragment or variant thereof that activates the CDK2 complex.Exemplary E7 fragment consists of a continuous amino terminal fragmentof E7 beginning at amino acid residue 1 and terminating at a carboxyterminal residue selected from the group consisting of amino acidresidues 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68 ,69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79 ,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,and 97 as set out in SEQ ID NO. 1. In a preferred embodiment, methods ofthe invention comprise use of an E7 fragment selected from the groupconsisting of amino acid residues 1 to 27, amino acid residues 1 to 38,amino acid residues 1 to 48, amino acid residues 1 to 69, and amino acidresidues 1 to 87 as set out in SEQ ID NO: 1. E7 variants as used hereininclude E7 proteins comprising additions, deletions, substitutions andother covalent modifications that result in a E7 polypeptide distinctfrom naturally occurring E7 but retaining the ability to induce CDK2kinase activity. Methods of the invention preferably include a CDK2substrate selected from the group consisting of histone H1, HPV proteinE1 and HPV protein E2, however methods of the invention also include useof any physiological, non-physiological, or synthetic substrate of CDK2.Synthetic substrates encompass non-naturally occurring fragments,analogs and variants of naturally occurring CDK2 substrates. Inpreferred embodiments, methods of the invention are performed in theabsence of CDK2 inhibitor proteins such as p21 and p27, therebyproviding for E7-induced kinase activity, rather than E7-inducedreduction in CDK2 inhibition.

[0016] The invention also provide methods for identifying an inhibitorof E7-induced CDK2 kinase activity comprising the steps of: a) measuringCDK2 kinase phosphorylation of a substrate; b) measuring increased CDK2kinase phosphorylation of a substrate in the presence of humanpapillomavirus (HPV) E7, or a fragment thereof, to determine E7-inducedCDK2 kinase activity; c) measuring CDK2 kinase phosphorylation of asubstrate in the presence of HPV E7, or a fragment thereof, and in thepresence of a test inhibitor compound; and d) identifying the testcompound as an inhibitor of E7-induced CDK2 kinase activity when theincreased phosphorylation measured in step (b) is reduced in step (c) inthe presence of the test compound. In one aspect, the methods of theinvention include use of an E7 fragment that activates CDK2 kinaseactivity wherein the E7 fragment consists of a continuous amino terminalfragment of E7 beginning at amino acid residue 1 and terminating at acarboxy terminal residue selected from the group consisting of aminoacid residues 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78 ,79 ,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, and 97 as set out in SEQ ID NO. 1. In a preferred embodiment,methods of the invention comprising use of an E7 fragment include an E7fragment selected from the group consisting of amino acid residues 1 to27, amino acid residues 1 to 38, amino acid residues 1 to 48, amino acidresidues 1 to 69, and amino acid residues 1 to 87 as set out in SEQ IDNO: 1. Methods of the invention preferably include a CDK2 substrateselected from the group consisting of histone H1, HPV protein E1 and HPVprotein E2. Components of the CDK2 complex which produces the CDK2kinase activity may include cyclins, e.g., members of the A familyincluding for example, A1 and A2, members of the B family including, forexample B1 and B2, cyclin C, members of the D family including forexample, D1, D2, and D3, members of the E family including for example,E1, E2 and Es, cyclin F, members of the G family including for example,G1 and G2, cyclin H, cyclin I, cyclin L, and members of the T familyincluding for example T1, T2a, and T2b to the extent that the cyclinbinds CDK2 and participates in effecting CDK2 kinase activity..

[0017] Methods of the invention comprehend use of CDK2 complex proteins,E7 and/or CDK2 substrate compounds from naturally occurring sources aswell as from recombinant sources transformed or transfected with one ormore polynucleotides encoding the desired recombinant product(s).Fragments of E7 that induce CDK2 kinase activity are also embraced.Preferably the CDK2 complex, E7 (or fragment thereof) and/or CDK2substrate compounds are recombinant products. The compounds can also beproduced by methods that facilitate purification, i.e., as fusionproteins comprising labels or tags thereby permitting production ofsignificantly pure compounds. Preferred labels or tags includeglutathione-S-transferase sequences (which permit purification usingglutathione agarose) or poly-histidine regions (permitting purificationusing nickel affinity chromatography). Other labels and tags well knownand routinely used in the art are also contemplated. The invention alsoembraces, however, methods employing crude preparations of E7, CDK2complex, and CDK2 substrate, but essentially free of other humanproteins.

[0018] Numerous embodiments of the methods of the invention are carriedout in order to detect a decrease in E7-induced CDK2 kinase activity inthe presence of a test compound that prevents, reverses, or destabilizesbinding between E7 (or a fragment thereof) and the CDK2 complex.Preferred methods of this type are performed in solution assays, andchanges in kinase activity are measured in the presence and absence of atest compound. Solution kinase assays of this type, e.g., histone kinaseassays, are well known and routinely practiced in the art.

[0019] Methods of the invention include those wherein either E7 (or afragment, variant, or analog thereof that retains the ability to induceCDK2 kinase activity) or bind the CDK2 complex is immobilized on a solidsupport and E7 (or a fragment, analog, or variant thereof) or the CDK2complex, whichever is not immobilized, is detectably labeled. Bindingbetween the two compounds is examined in the presence and absence of atest compound and a change in the amount of bound detectable label ismeasured. In assays wherein a lower amount of bound label is detected inthe presence of the test compound, the test compound is identified as aninhibitor of E7 binding to the CDK2 complex. Subsequent kinase assayscan be employed to determine the effect of the test compound on kinaseactivity.

[0020] The invention also comprehends cell-based assays whereininhibitors of E7 and CDK2 complex binding can be identified, as well asinhibitors of E7-enhanced phosphorylation. In one aspect, the inventionprovides split hybrid assays as generally described in WO 98/13502,published Apr. 2, 1998, incorporated by reference herein, wherein, forexample, (i) components of the CDK2 complex are expressed as a fusionprotein with amino acid sequences comprising either a transcriptionfactor DNA binding domain or a transcription factor transactivatingdomain, and (ii) E7 (or a fragment thereof) is also expressed as afusion protein with whichever transcription factor domain is not fusedto the CDK2 complex protein. Binding between the CDK2 complex and E7fusion proteins brings into proximity the two components of thetranscription factor to produce a biologically active transcriptionfactor. A plasmid encoding a repressor gene is also introduced into thesame cell type and expression of the repressor is driven by atranscription element recognized by the biologically activetranscription factor comprising the two fusion proteins. The expressedrepressor then acts to prevent transcription of a reporter gene. In thepresence of a compound that inhibits binding between the E7 and CDK2complex fusion proteins, the biologically active transcription factor isnot formed, the repressor protein is not expressed, and transcription ofthe reporter gene is permitted. In assays of this type, inhibition of aspecific binding interaction is detected by a positive signal, i.e.,expression of a detectable reporter gene. The invention also embracesnumerous variations on this method as described in WO 95/20652,published Aug. 3, 1995, incorporated by reference herein. In addition,the invention embraces di-hybrid, or two-hybrid assays as previouslydescribed [Fields and Song, Nature 340:245-246 (1989); Fields, Methods:A Companion to Methods in Enzymology 5:116-124 (1993); U.S. Pat.5,283,173 issued Feb. 1, 1994 to Fields, et al.], wherein inhibition ofa specific binding interaction is detected by a negative signal, i.e.,loss or expression of a reporter gene. Modifications and variations onthe di-hybrid assay (also referred to in the art as “two-hybrid” assays)have previously been described [Colas and Brent, TIBTECH 16:355-363(1998)] and are embraced by the invention. In a cell-based assay,however, detection of inhibition of binding with a positive signal ispreferable.

[0021] In another aspect, cell based assays are provided wherein E7 andcomponents of the CDK2 kinase complex are expressed (or overexpressed)in a cell and inhibitors identified which decrease E7-enhancedphosphorylation. Optionally, detection of E7-enhanced phosphorylationcan be accomplished using a control cell line which expresses CDK2kinase complex components in the absence of E7. Kinase activity in thiscontrol cell would identify the basal level of kinase complex activity.Cell lines for assays of this type may be produced through introductionof exogenous DNA encoding E7 and/or components of the CDK2 complex orintroducing exogenous DNA which increases expression of one or more ofthese assay component which are encoded in the endogenous cellulargenome.

[0022] Assays of the invention are particularly amenable to highthroughput screening (HTS) assays. HTS permit screening of large numbers(i.e., tens to thousands or more) of compounds in an efficient manner.Cell-based HTS systems are also embraced, including melanophore assays,yeast-based assay systems, and mammalian cell expression systems[Jayawickreme and Kost, Curr. Opin. Biotechnol. 8:629-634 (1997)].Automated and miniaturized HTS assays are particularly preferred[Houston and Banks, Curr. Opin. Biotechnol. 8:734-740 (1997)]. HTSassays are designed to identify “hits” or “lead compounds” having thedesired inhibitory property, from which modifications can be designed toimprove the desired property. Chemical modification of the “hit” or“lead compound” is often based on an identifiable structure/activityrelationship between the “hit” and one or more of the binding partnerpolypeptides.

[0023] There are a number of different libraries used for theidentification of specific small molecule inhibitors, including, (1)chemical libraries, (2) natural product libraries, and (3) combinatoriallibraries comprised of random peptides, oligonucleotides or organicmolecules.

[0024] Chemical libraries consist of structural analogs of knowncompounds or compounds that are identified as “hits” or “leads” vianatural product screening. Natural product libraries are derived fromcollections of microorganisms, animals, plants, or marine organismswhich are used to create mixtures for screening by: (1) fermentation andextraction of broths from soil, plant or marine microorganisms or (2)extraction of plants or marine organisms. Natural product librariesinclude polyketides, non-ribosomal peptides, and variants (non-naturallyoccurring) thereof. For a review, see Science 282:63-68 (1998),incorporated by reference herein. Combinatorial libraries are composedof large numbers of peptides, oligonucleotides or organic compounds as amixture. They are relatively easy to prepare by traditional automatedsynthesis methods, PCR, cloning or proprietary synthetic methods. Ofparticular interest are peptide and oligonucleotide combinatoriallibraries. Still other libraries of interest include peptide, protein,peptidomimetic, multiparallel synthetic collection, recombinatorial, andpolypeptide libraries. For a review of combinatorial chemistry andlibraries created therefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997), incorporated by reference herein.

[0025] The invention further provides specific inhibitors identified bythe methods of the invention. Specific inhibitors are defined as thosethat act to preclude, reverse or disrupt E7 binding to CDK2 complex (orCDK2 kinase activity), and/or prevent, reverse or disrupt E7-inducedincrease in CDK2 kinase activity. Compositions comprising a specificinhibitor of E7-induced CDK2 kinase activity and a pharmaceuticallyacceptable carrier are also provided. Preferably, compositions of theinvention are pharmaceutical compositions. The invention also providesuse of an inhibitor of E7 binding to the CDK2 complex in the productionof a medicament for ameliorating viral infection, e.g., HPV infection,adenoviral infection, or SV40 infection..

[0026] The pharmaceutical compositions of the invention optionally mayinclude pharmaceutically acceptable (i.e., sterile and non-toxic)liquid, semisolid, or solid diluents that serve as pharmaceuticalvehicles, excipients, or media. Any diluent known in the art may beused. Exemplary diluents include, but are not limited to,polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- andpropylhydroxybenzoate, talc, alginates, starches, lactose, sucrose,dextrose, sorbitol, mannitol, gum acacia, calcium phosphate, mineraloil, cocoa butter, and oil of theobroma.

[0027] The pharmaceutical compositions can be packaged in formsconvenient for delivery. The compositions can be enclosed within acapsule, sachet, cachet, gelatin, paper, or other container. Thesedelivery forms are preferred when compatible with entry of thecomposition into the recipient organism and, particularly, when thecomposition is being delivered in unit dose form. The dosage units canbe packaged, e.g., in tablets, capsules, suppositories or cachets.

[0028] The invention also provides methods for identifying an anti-viralagent comprising the steps of: a) identifying an inhibitor of E7-inducedincrease in CDK2 kinase activity; b) measuring viral proliferation inthe presence and absence of the inhibitor identified in (a); and c)identifying the inhibitor as an antiviral agent when decreased viralproliferation is detected in the presence of the inhibitor compared toviral proliferation in the absence of the inhibitor. In one aspect,methods to identify an inhibitor of E7-induced CDK2 kinase activity areutilized to screen test compounds for use as antiviral agents. Theinhibitors identified are utilized in assays that permit determinationof the inhibitor's ability to act as an anti-viral agent in, forexample, cell-based assays (i.e., cell culture systems) and/or animalmodels of HPV viral infection.

[0029] Another aspect of the invention provides methods for inhibitingE7-induced CDK2 kinase activity comprising the steps of administering toan individual in need thereof an effective amount of an inhibitor ofE7-induced CKD2 kinase activity or an inhibitor of E7 binding to theCDK2 complex. Also provided are methods for ameliorating (inhibiting)viral proliferation comprising the steps of administering to anindividual in need thereof an effective amount of an inhibitor ofE7-induced CKD2 kinase activity or an inhibitor or E7 binding to theCDK2 complex. The invention further provides methods for preventing ortreating viral infection comprising the steps of administering to anindividual in need thereof an effective amount of an inhibitor ofE7-induced CKD2 kinase activity or an inhibitor of E7 binding to theCDK2 complex. Preferably, the individual in need thereof is administereda pharmaceutical composition of the invention. The pharmaceuticalcompositions may be introduced into the subject to be treated by anyconventional method including, e.g., by intravenous, intradermal,intramuscular, intramammary, intraperitoneal, intrathecal, intraocular,retrobulbar, intrapulmonary (e.g., aerosolized drug solutions) orsubcutaneous injection (including depot administration for long termrelease); by oral, sublingual, nasal, anal, vaginal, or transdermaldelivery; or by surgical implantation, e.g., embedded under the spleniccapsule, brain, or in the cornea. The treatment may consist of a singledose or a plurality of doses over a period of time. Co-administration ofother anti-viral agents including, e.g., acyclovir, gancyclovir,vidarabidine, foscarnet, cidofovir, amantidine, ribavirin,trifluorothymidine, interferon-α, zidovudine, didanosine or zalcitabine,is also contemplated.

[0030] When given parenterally, specific binding inhibitor compositionsare generally injected in doses ranging from 1 μg/kg to 100 mg/kg perday, preferably at doses ranging from 0.1 mg/kg to 50 mg/kg per day, andmore preferably at doses ranging from 1 to 20 mg/kg/day. The inhibitorcomposition may be administered by an initial bolus followed by acontinuous infusion to maintain therapeutic circulating levels of a drugproduct. Those of ordinary skill in the art will readily optimizeeffective dosages and administration regimens as determined by goodmedical practice and the clinical condition of the individual patient.The frequency of dosing will depend on the pharmacokinetic parameters ofthe agents and the route of administration. The optimal pharmaceuticalformulation will be determined by one skilled in the art depending uponthe route of administration and desired dosage. See for example,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa. 18042) pages 1435-1712, the disclosure of which ishereby incorporated by reference. Such formulations may influence thephysical state, stability, rate of in vivo release, and rate of in vivoclearance of the administered agents. Depending on the route ofadministration, a suitable dose may be calculated according to bodyweight, body surface area, or organ size. Further refinement of thecalculations necessary to determine the appropriate dosage for treatmentinvolving each of the above-mentioned formulations is routinely made bythose of ordinary skill in the art without undue experimentation,especially in light of the dosage information and assays disclosedherein, as well as the pharmacokinetic data observed in human clinicaltrials. Appropriate dosages may be ascertained through use ofestablished assays for determining blood levels dosages in conjunctionwith appropriate dose-response data. The final dosage regimen will bedetermined by the attending physician, considering various factors whichmodify the action of drugs, e.g., the drug's specific activity, theseverity of the damage and the responsiveness of the patient, the age,condition, body weight, sex and diet of the patient, the severity of anyinfection, time of administration and other clinical factors. As studiesare conducted, further information will emerge regarding the appropriatedosage levels and duration of treatment for various diseases andconditions.

[0031] It will be appreciated that the pharmaceutical compositions andtreatment methods of the invention may be useful in the fields of humanmedicine and veterinary medicine. Thus, the subject to be treated may bea mammal, preferably human, or other animals. For veterinary purposes,subjects include, for example, farm animals including cows, sheep, pigs,horses, and goats, companion animals such as dogs and cats; exoticand/or zoo animals; laboratory animals including mice, rats, rabbits,guinea pigs, and hamsters; and poultry such as chickens, turkeys, ducksand geese.

EXAMPLES

[0032] The invention is illustrated by the following examples. Example 1relates binding assays that characterize E7 interactions with variousproteins. Example 2 describes kinase assays wherein the effect of E7 onCDK2 activity examined. Example 3 relates to the effects of HPV E7 onvarious kinases. Example 4 describes kinetics of E7-induced activationof CDK2. Example 5 addresses identification of E7 amino acid residuesrequired for CDK2 activation. Example 6 describes interaction of E7 withcyclina.

Example 1 E7 Binding Assays

[0033] In an attempt to characterize HPV E7 binding interactions, assayswere designed using either HPV6b E7, HPV16 E7 or HPV31 E7 and variouscellular proteins or fragments thereof. The E7 proteins were expressedas glutathione-S-transferase (GST) fusion proteins in order tofacilitate purification and expression constructs were prepared asfollows.

[0034] E7 Expression Constructs

[0035] The E7 gene was amplified by polymerase chain reaction (PCR) fromcloned HPV31, HPV16, and HPV6b DNA (American Type Culture Collection[ATCC] for HPV 31 and HPV 6b; Seedoffet al., Virol. 145:181-185 (1985)for HPV 16, also available from the ATCC). PCR primers, as set outbelow, were designed to engineer BamHI or XhoI sites in theamplification product to facilitate in-frame subcloning into expressionvector pGEX-4T-3 (Pharmacia Biotech). The vector was used to express theproduct of each subcloned sequence as a fusion protein with aglutathione-S-transferase (GST) moiety at the amino-terminus.

[0036] HPV31 primers

[0037] E7 Sense (with a BamHI site) SEQ ID NO: 2

[0038] CGGGATCCATGCGTGGAGAAACACCTAC

[0039] E7 Antisense (with a BamHI site) SEQ ID NO: 3

[0040] CGGGATCCTTACAGTCTAGTAGAACAG

[0041] HPV 6b primers

[0042] E7 Sense (with a BamHI site) SEQ ID NO: 4

[0043] CGGGATCCATGCATGGAAGACATGTT

[0044] E7-Antisense (with an XhoI site) SEQ ID NO: 5

[0045] CCGCTCGAGTTAGGTCTTCGGTGCGC

[0046] HPV 16 primers

[0047] E7 Sense (with a BamHI site) SEQ ID NO: 6

[0048] CGGGATCCATGCATGGAGATACACCTAC

[0049] E7 Antisense (with an XhoI site) SEQ ID NO: 7

[0050] CCGCTCGAGTTATGGTTTCTGAGAACAGATG

[0051] For HPV31E7, the amplification product was subcloned intopGEX-4T-3 previously digested with BamHI, while HPV6b and HPV16 E7amplification products were subcloned into pGEX-4T-3 previously digestedwith BamHI and XhoI. Proper orientation was determined using restrictionmapping, sequence analysis was carried out using Perkin-Elmer ABI-Prism®technology, and the expression constructs were individually transfectedinto a JM 109 strain of E. coli. Bacteria in log-growth phase wereharvested by centrifugation after a four hour induction with 0.5 mM IPTGat 30° C. The cell pellet was resuspended in lysis buffer containing11.3 mM NaH₂PO₄, 38.7 mM Na₂HPO₄, 0.07% β-mercaptoethanol, 10 mM EDTA,0.5 mM PMSF, 0.025 IU/ml aprotinin, and 10 nM leupeptin, and protein waspurified using glutathione-agarose beads (Sigma) according tomanufacturer's suggested protocol. After extensive washing with PBS, theGST-E7-coupled beads were stored at 4° C. until use. Protein levels weremeasured using a BioRad protein assay kit, and purified proteinpreparations were examined by SDS-PAGE followed by Coomassie bluestaining and Western blotting.

[0052] Binding Protein Expression Constructs

[0053] Full length E7-binding proteins p21 and p27 were expressed withpoly-histidine tags to facilitate purification. Histidine-taggedfragments of p21 and p27 were also expressed comprising amino terminalresidues 1-78 of p21 (designated His-p21N), carboxy terminal residues72-164 of p21 (designated His-p21C), amino terminal residues 1-101 fromp27 (designated His-p27N) or carboxy terminal residues 95-198 of p27(designated His-p27C). Expression constructs encoding the full lengthproteins or fragment polypeptides were prepared as follows.

[0054] The full-length, histidine-labeled p21 protein, designatedHis-p21, was subcloned as a XhoI/BamHI PCR fragment into pET15b(Novagen) previously digested with the same two enzymes. Template DNAwas the cloned p²¹ coding sequence [Harper et al., Cell 75:805-816(1993)] and primers included p-21-1′ and p21-4′ to amplify the fulllength p21 coding region. The p21 amino and carboxy terminalhistidine-tagged fragments were also amplified by PCR to produceXhoI/BamHI fragments using primers p21-1′ and p21-2′ to amplifysequences encoding residues 1 to 78, and p21-3′ and p21-4′ to amplifythe region encoding residues 72 to 164.

[0055] p21-1′ GCCTCGAGATGTCAGAACCGGCTGGGGATG SEQ ID NO: 8

[0056] p21-2′ GCGGATCCTTAGAAGGTAGAGCTTGGGCAG SEQ ID NO: 9

[0057] p21-3′ GCCTCGAGCTGCCCAAGCTCTACCTTC SEQ ID NO: 10

[0058] p21-4′ GCGGATCCTTAGGGCTTCCTCTTGGAG SEQ ID NO: 12

[0059] Amplification reactions were carried out using Ready-to-Go™ PCRReaction Beads (Amersham-Pharmacia Biotech) according to themanufacturer's suggested protocol Reaction products were digested withBamHI and XhoI, extracted with equal volumes of phenol and chloroform,precipitated with ethanol, and suspended in 20 μl water. Ligation intovector pET15b (Novagen) was carried out overnight at 15° C. in areaction containing 5 μl PCR amplification product, 2 μl vector DNA(previously digested with BamHI and XhoI, 2 μl 5× ligation buffer (BRL)and 1 μl T4 ligase. Clones having proper orientation were selected andtheir sequences confirmed by restriction analysis and PCR sequencingusing ABI-PRISM®® (PE Applied Biosystems) technology.

[0060] Similarly, the histidine labeled, full-length p27 protein,designated His-p27, was subcloned as a XhoI/BamHI PCR fragment intopET15b (Novagen) previously digested with the same two enzymes. DNAencoding human p27 [Toyoshima and Hunter, Cell 78:67-74 (1994)] was usedas template in an amplification reaction with primer pairs p27-1 andp27-5 to amplify the full length p27 cDNA. The same template DNA wasused to amplify polynucleotides encoding the histidine-labeled amino andcarboxy terminal fragments of p27, with primer pair p27-7 and p27-8 usedto amplify DNA encoding p27 residues 1 to 101, and primer pair p27-9 andp27-10 used to generate a DNA encoding residues 95 to 198.

[0061] p27-1 CGGATCCTATGTCAAACGTGCGAGTG SEQ ID NO: 11

[0062] p27-5 AGGATCCTTACGTTTGACGTCTTCTG SEQ ID NO: 13

[0063] p27-7′ GCCTCGAGATGTCAAACGTGCGAGTGTC SEQ ID NO: 14

[0064] p-27-8′ GCGGATCCTTACACCTTGCAGGCACCTTTG SEQ ID NO: 15

[0065] p27-9′ GCCTCGAGAAAGGTGCCTGCAAGGTGC SEQ ID NO: 16

[0066] p27-10 GCGGATCCTTACGTTTGACGTCTCTG SEQ ID NO: 17

[0067] All PCR amplifications were carried out using Ready-to-Go™ PCRReaction Beads (Amersham-Pharmacia Biotech) according to themanufacturer's suggested protocol.

[0068] In producing the full length p27 expression vector, the PCRamplification product was first subcloned into vector pCRII using a TAcloning kit (Invitrogen) according to the manufacturer's suggestedprotocol. The full length cDNA was removed from the vector with BamHIdigestion and the sequence isolated using 1% agarose gelelectrophoresis. The full length band was excised from the gel and thefragment purified using a GeneClean® kit (Bio101) according to themanufacturer's suggested protocol. The isolated fragment was subclonedinto vector pTrcHisB (Invitrogen) previously digested with BamHI, andligation was carried out using 5 μl digested PCR product, 2 μl vector, 2μl 5× ligation buffer (BRL), and 1 μl T4 ligase (BRL). The ligationreaction was used to transform JM 109 competent cells according tostandard transformation protocols.

[0069] For cloning p27 DNA fragments encoding residues 1 to 101 and 95to 198, PCR products were digested with BamHI and XhoI, extracted withequal volumes of phenol and chloroform, precipitated with ethanol, andresuspended in water. Ligation was carried out as described above, andthe ligation products were used to transform E. coli strain DH5α. Cloneswere selected and orientation determined by restriction analysis and PCRsequencing using ABI-PRISM® technology.

[0070] Expression constructs encoding the histidine-tagged proteins weretransformed into E. coli strain BL21(DE3) using standard techniques andexpressed proteins were purified using nickel agarose affinitychromatography under denaturing conditions. Eluted proteins wererefolded by extensive, step-wise dialysis using techniques well known inthe art.

[0071] Preparation of HEK293 Cell Lysate

[0072] HEK293 cells were collected by centrifugation, washed twice inphosphate buffered saline, resuspended in three volumes of low saltbuffer containing 20 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM sodiumvanadate, 1 mM EDTA, 1 mM EGTA, 0.1% NP-40 and 10% glycerol, andincubated on ice for 10 minutes. Cells were centrifuged at 13,000×g andsupernatant containing the cytoplasmic extract was collected. Thepellet, containing the nuclei, was washed once in the low salt buffer,and suspended in three volumes of a high salt buffer containing 20 mMHEPES, pH 7.9, 420 mM NaCl, 10 mM KCl, 0.1 mM sodium vanadate, 1 mMEDTA, 1 mM EGTA, 0.1% NP-40 and 10% glycerol. The nuclear suspension wasincubated with agitation at 4° C. and centrifuged at 13,000×g, afterwhich the nuclear lysate was collected and stored until use. Inprecipitation assays (described below), nuclear extract was mixed withcytoplasmic extract at a ratio of 1:2.

[0073] Binding Assay Conditions

[0074] GST-16E7 or GST-31E7 fusion proteins bound to glutathione-agarosebeads were incubated with either purified pRb (QED Bioscience Inc.),His-p21, His-p21N, His-p21C, His-p27, His-p27N, His-p27C, or HEK293 celllysate at 4° C. for two hours in binding buffer containing 20 mM HEPES,pH 7.4, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 140 mM NaCl, 13% glycerol, 1 mMDTT, and 1× protease inhibitor tablet (Boehringer Mannheim). Afterwashing five times with binding buffer, the glutathione-agarose beadswere resuspended in SDS-sample buffer and heated to 100° C. to elutebinding proteins. Elited proteins were separated using 15% SDS-PAGE andthe proteins were analyzed by Coomassie blue staining and Westernblotting.

[0075] Results indicated that GST-31E7 bound purified pRb as well as pRbin HEK 293 cell lysate. E7 proteins from the HPV strains were found tobind recombinant forms of both p21 and p27, consistent with previousobservations [Funk, et al., Genes & Dev 11:2090-100 (1997); Jones, etal., Genes & Dev 11:2101-11 (1997), Zerfass, et al., Oncogene 13:2323-30(1996)]. However, neither amino- or carboxy-terminal fragments of p21 orp27 were able to significantly bind E7 relative to the full lengthmolecule, suggesting that neither the carboxy- or amino-terminal halvesof p21 or p27 are sufficient for E7 binding.

Example 2 Kinase Assays

[0076] Kinase assays were performed with CDK2 co-expressed with cyclin Eor cyclin A from baculovirus vectors in Sf9 cells as described below.

[0077] Baculovirus Vector Construction

[0078] Recombinant cyclin E and cyclin A was prepared as follows. DNAencoding cyclin E was cloned from human liver cDNA using primers KW133and PH 113 designed based on the published sequence.

[0079] KW133 GATCAGATCTCATGAAGGAGGACGGCGGCG SEQ ID NO: 18

[0080] RH113 GCAGATCTTCAGTGGTGGTGGTGGTGGTGG SEQ ID NO: 19

[0081] The cyclin E amplification product was modified in the PCR tointroduce aberrant nucleotides with the 3′ RH113 primer producing ahyperstable form of the protein. The amplification product was insertedinto vector pAcGHLT-C (PharMingen) which expresses encoded proteins as aGST fusion.

[0082] The cyclin A gene [Pines and Hunter, Nature 346:760-763 (1990)]was amplified by PCR from using primers RH101 and RH103 that introducedNotI restriction sites flanking the fusion gene amplification product.

[0083] RH102 GGGCGGCCGCATGTCCCCTATACTAGGTTAT SEQ ID NO: 20

[0084] RH103 GGGCGGCCGCGTCAGTCAGTCACGATG SEQ ID NO: 21

[0085] The amplification product was subcloned into baculovirus transfervector pVL1392 (PharMingen) which expresses encoded proteins as GSTfusions. Restriction mapping identified clones with correct insertorientation, and one clone, pVL-GST-cyclin A, was sequenced and verifiedto be wild-type.

[0086] Baculovirus Expression

[0087] Recombinant baculoviruses were produced by co-transfection ofSpodoptera frugiperda cells (Sf9) with cyclin transfer vectors andBaculoGold crippled baculoviral DNA (PharMingen) according tomanufacturer's suggested protocol. Resultant viruses were purifiedthrough three consecutive plaque purifications. A baculovirus expressingCDK2 with a hemagglutinin tag as previously described (Desai et al.,Mol. Biol. Cell 3: 571-582) was used for CDK2 expression.

[0088] Purification of Cyclin/CDK2 Complexes

[0089] Cyclin E and cyclin A were co-expressed with CDK2 by co-infectionof either Sf9 or High Five™ (Invitrogen) cells with the recombinantbaculoviruses at a multiplicity of infection (m.o.i.) of 20 for thecyclin-expressing viruses and 10 for the CDK2-expressing virus. Sf9cells were grown at 28° C. in spinner flasks in Grace's medium(GIBCO/BRL) containing 10% fetal bovine serum (FBS), 100 units/mlpenicillin, and 100 μg/ml streptomycin. High Five cells were grown at28° C. in shaker flasks at 150 r.p.m. in InsectEXPRESS™ medium (BioWhittaker).

[0090] Cells were collected by centrifugation, cell pellets were washedwith phosphate buffered saline (PBS), and the cells were resuspended inhypotonic lysis buffer containing 10 mM HEPES, pH 7.4, 10 mM NaCl, 1 mMEDTA, 0.2 μg/ml leupeptin, 0.2 μg/ml pepstatin, 0.2 μg/ml aprotinin, and0.2 mM AEBSF at a density of 1 ml for each 10⁷ cells initially infected.After an hour, NaCl was added to a final concentration of 150 mM. Cellswere sonicated on ice for two minutes (100% duty cycle with an output of4) and the lysate was clarified by centrifugation. The GST-cyclin/CDK2complexes were isolated by affinity chromatography usingglutathione-Sepharose® beads (Pharmacia Biotech). The beads were washedextensively with PBS and the complexes eluted with 15 mM reducedglutathione in 50 mM Tris-HCl, pH 8.0. Eluted protein was analyzed bySDS-PAGE and Western blotting to confirm purity of the cyclin A- andcyclin E-CDK2 complexes.

[0091] MBP-E1 and MBP-E2 Expression and Purification

[0092] A maltose-binding protein (MBP)-E1 fusion protein expressionvector was constructed as follows. Briefly, the vector was constructedby amplifying the HPV18 E1 gene from pSGE1-18 [Sverdrup and Khan, J.Virol. 68:505-509 (1994)] and cloning the resulting amplificationproduct into pMAL-CR1 (New England Biolabs, Inc.) previously digestedwith BamHI. Primers to the 5′ and 3′ ends of the E1 gene incorporatedBamHI or BglII sites into the amplification product. The resultingvector encoded an inducible MBP-E1 fusion protein.

[0093] The MBP-E1 fusion protein was purified according to themanufacturer's suggested protocol. Briefly, E. coli strain DH5expressing recombinant MBP-E1 fusion protein was fermented at the fourliter scale. Cells were collected by centrifugation, the cell pellet wasresuspended in column buffer containing 20 mM HEPES, pH 7.4, 200 mMNaCl, 1 mM EDTA, 10% glycerol, and 5 mM β-mercaptoethanol, and the cellswere lysed using an Avestin Emulsiflex™ dynamic homogenizer at 15,000 to20,000 psi. The lysate was centrifuged at 25,000×g for 60 minutes at 5to 8° C., and the supernatant was filtered through a 0.45 μm filter. Thefiltered supernatant was loaded onto a 2.6 cm amylose-affinity columncontaining 50 ml amylose resin (New England BioLabs). The column waswashed with 12 bed volumes of column buffer and eluted using 10 mMmaltose in column buffer. Eluate fractions were analyzed for purityusing SDS-PAGE.

[0094] A construct encoding an E2 maltose-binding fusion protein wasprepared in a similar manner.

[0095] Kinase Assay

[0096] Histone H1 was chosen as the substrate in the kinase assays sincepRb, a physiological CDK2 substrate, is known to bind E7 and couldinterfere with the assay. When indicated, His-p27 was added at aconcentration previously demonstrated to inhibit 50% of the CDK2 kinaseactivity.

[0097] GST-E7 fusion proteins were prepared as described above andeluted from the glutathione-agarose beads using buffer containing 50 mMTris-HCl (pH 8.0), 5 mM reduced glutathione, 0.1% Triton® X-100, 1 mMDTT, and 1× protease inhibitor tablet (Boehringer Mannheim).

[0098] For some assays, HEK293 cell lysate (100 μg total protein,prepared as described above) was preincubated with purified GST-31E7protein in PBS in the presence of p13 agarose beads (Calbiochem) at 4°C. for one hour, and subsequently incubated with purified His-p27 for 30min at 4° C. Preincubation was carried out to permit protein complexformation, while p13 agarose beads bind cyclin-dependent kinasecomplexes to facilitate purification [Dreatta, et al., Cell 56:829-838(1989)]. After collection and washing four times with PBS, the beadswere washed two additional times with KIP buffer containing 50 mMTris-Cl, pH 7.5, 10 mM MgCl₂, 5 mM EGTA, 2 mM DTT, and 10 μM ATP. Thebeads were resuspended in 35 μl KIP buffer, mixed with 35 μl histonekinase buffer containing 0.6 mg/ml histone H1, 0.2 mM ATP, 5 μCi[γ-³²P]-ATP/assay, 40 mM HEPES, pH 7.3, 10 mM EGTA, and 20 MM MgCl₂, andincubated at room temperature for 20 minute. The reaction was stopped byadding 70 μl of 2× SDS-sample buffer. Phosphorylated proteins wereanalyzed by SDS-PAGE followed by autoradiography and PhosphorImager®analysis.

[0099] In other assays, purified cyclin A- or cyclin E-complexed CDK2was included. GST-E7 was added directly to purified recombinantcyclin/CDK2 complex in kinase buffer (20 mM MgCl, 10 mM EGTA, and 40 mMHEPES, pH 7.0) and incubated on ice for 20 min prior to beginning thekinase assay. In some assays, GST-E7 was preincubated with p21 or p27.Kinase activity was measured following addition of 1 μl of 2 mCi/ml[γ-³²P] ATP and 3.75 μg histone H1 per 30 μl assay and incubation at 30°C. for 20 min. The reaction was stopped by adding SDS sample buffer, andphosphorylated proteins were analyzed by SDS-PAGE followed byautoradiography and PhosphorImager® analysis.

[0100] Results indicated that E7 had little or no effect on p21- andp27-dependent inhibition of cyclin E/CDK2 activity, and theseobservations were inconsistent with previous reports that E7significantly blocks p21 [Funk, et al., Genes & Dev 11:2090-100 (1997);Jones, et al., Genes & Dev 11:2101-11 (1997)], and/or p27 [Zerfass, etal., Oncogene 13:2323-30 (1996)] inhibition activity. At best, only asmall or no effect on the inhibitory activity of p27 was noted.

[0101] Purified cyclin E/CDK2 and cyclin A/CDK2 were used to directlyexamine the effects of p27 and E7 in a solution kinase assay. Similar tothe above results, little or no effect of GST-E7 on p27 inhibition ofCDK2 activity was noted. However, a significant effect of GST-16E7 andGST-31E7 on cyclin E/CDK2 and cyclin A/CDK2 histone H1 kinase activitywas noted. PhosphorImager® analysis indicated a 26-fold increase incyclinA/CDK2 kinase activity in the presence of GST-16E7. A smaller1.9-fold increase in pRb kinase activity was also noted. No kinaseactivity was noted in any GST-E7 preparation and GST alone also had noeffect. These results indicated that E7 was able to significantly andsubstantially alter the substrate specificity of cyclin/CDK complexes. Alinear dose response was noted for the GST-E7 effect, with a 50%stimulatory concentration of E7 of approximately 0.18 μM and a peakstimulatory activity of approximately 0.29 μM. These results suggestedthat E7 was able to directly alter CDK2 activity and/or substratespecificity using histone H1 protein as substrate.

[0102] Results also indicated that cyclin E/CDK2 phosphorylation ofpurified MBP-HPV 18 E1 fusion protein was stimulated 2.3-fold by theaddition of HPV 16E7. CDK2 did not phosphorylate MBP alone. Thephosphorylation of E1 was completely inhibited by the addition of p27even in the presence of excess GST-16E7. Thus, GST-E7 was able todirectly promote CDK2-dependent phosphorylation of multiple substrates,including HPV18 E1. Phosphorylation of HPV E2 by CDK2/cyclinA was alsoenhanced by addition of E7, but E2 was a poor substrate for CDK2/cyclinEin the presence or absence of E7.

[0103] E1 phosphorylation is an important biological event because itpermits amplification of the HPV genome in differentiated cells thatwould not normally support DNA synthesis. E1 is a 72 kDa phosphoproteinthat initiates ori-dependent HPV replication [Cho, et al., Intervirology1994;37:150-8 (1994)]. Based on amino acid sequence similarity, E1 hasbeen grouped in the helicase superfamily III [Koonin, Nucl. Acids Res.21:2541-7 (1993)]. Phosphorylation of E1 is important for activating HPVDNA replicating activity in vitro, suggesting that phosphorylation isimportant for coordinating viral and host replication machinery [Ma, etal., Proc. Natl. Acad. Sci. (USA) 96:382-7 (1999)]. E1 possesses an RXLcyclin-interaction motif which enables its efficient phosphorylation bycyclin-dependent kinases (CDKs). E1 lacking CDK phosphorylation sitesbinds E2 but is deficient for activating HPV replication [Ma, et al.,Proc. Natl. Acad. Sci. (USA) 96:382-7 (1999)]. The phosphorylationresults described above therefore suggest a mechanism by which HPV E7might redirect CDK2 specificity to E1, and suggest a means by which E7might preferentially promote viral DNA replication in differentiatedkeratinocytes.

[0104] Results also suggested that E7 can bind p21 and p27, but that E7has little or no ability to overcome the CDK inhibitory activity ofthese proteins, and these results are inconsistent with previousreports. Previously published work, however, used immunoprecipitatedkinase activity for studying functional aspects of E7-CDK inhibitorinteraction [Funk, et al., Genes & Dev 11:2090-100 (1997); Jones, etal., Genes & Dev 11:2101-11 (1997), Zerfass, et al., Oncogene 13:2323-30(1996)] while the present studies utilized purified kinase complex. Theprevious studies may have therefore overlooked any direct E7 effects onkinase activity, or misinterpreted such effects as arising from bindingto the inhibitor rather than binding directly to the kinase, due to thelack of purity of these preparations.

[0105] In addition, the present results indicated that HPV-E7 derivedfrom papillomaviruses with both high or low potential for oncogenicprogression significantly activated CDK2. HPV31 and HPV16 activated CDK2histone kinase activity by 12.6- and 18.2-fold, respectively, while thelow risk-derived E7 from HPV6b activated by 15.8 fold.

Example 3 Effects of HPV E7 On Various Kinases

[0106] In an attempt to determine if the E7 effects on kinase activitywere specific for members of the cyclin-dependent kinase family, variouskinase/substrate combinations were examined for enhanced activity in thepresence of E7.

[0107] When HPV16-E7 was added to either cyclinA/CDK2 or cyclinE/CDK2using histoneH1 as the substrate, a large increase (26-fold and 13-fold)over controls was noted as described above. When the retinoblastoma(pRb) protein was used as a substrate for cyclinE/CDK2 a 1.9-foldincrease over background was noted. The related cyclin-dependent kinaseCDK5 complexed with p25 also showed an increase in H1 phosphorylation,albeit less than CDK2, with a 4.5-fold increase over controls. In otherassays, it was observed that MAP kinase I activity and CDC2/cyclinBactivity were unaffected by HPV16 E7, suggesting that E7 kinaseactivation is specific for members of the cyclin-dependent kinasefamily.

Example 4 Kinetics of Activation of CDK2 by HPV E7

[0108] Experiments were then designed to determine the effects ofsubstrate concentration on HPV16-E7 stimulation of CDK2/cyclinA histonekinase activity using the kinase assay experimental conditions describedabove in Example 2 with minor modifications. Initial experimentsindicated that a 15 minute assay fell within the linear range of thekinase activity, and as a result, all assays were carried out for 15minutes. In addition, cyclinA/CDK2 complex was pre-incubated with anamount of HPV16-E7 that had been determined in previous experiments toyield the maximum activation of CDK2 kinase.

[0109] Using this approach, results permitted a non-linear least squarescurve fit using the Michaelis-Menton equation to give a K_(m) of1.1+/−0.2 μM for histone H1, a concentration similar to the K_(m) of 1.4μM previously determined for histone phosphorylation by cyclinA/CDK2[Connell-Crowley et al., Mol. Biol. Cell 4: 79-92, 1993]. These resultstherefore suggested that the large effect that HPV-E7 has upon CDK2activity was not achieved through alteration of substrate binding, butinstead through direct alteration of the CDK2 catalytic mechanism.

Example 5 Mutagenesis of E7 and Determination of Critical Amino AcidSequences

[0110] In an attempt to identify E7 amino acids critical to CDK2 bindingand/or activation, point mutations were introduced into the E7 sequenceand changes in CDK2 kinase activation were determined as [follows]discussed below. In general, the structure of E7 can be divided intothree regions: CR1 extends from amino acid residue 1 to approximatelyresidue 16, CR2 includes approximately amino acids 17 to 38, and CR3encompasses approximately residue 39 to 98. Within each of CR2 and CR3,several biologically significant regions have been identified,including, a Rb binding domain (residues 22-26) and casein kinase IIphosphorylation site (residues 31-37) in CR2, and at least twozinc-binding domains in CR3. The various mutants were prepared in viewof these E7 regions.

[0111] E7 Point Mutations

[0112] All site directed mutations of E7 constructs were made using aQuikChange™ kit (Stratagene). In most cases, HPV16 E7 was mutated. Thefollowing conservative amino acid changes within previously definedfunctional regions of the 98 amino acid protein HPV 16 E7 wereintroduced: Amino Acid Changes E7 Functional Region M12→L12 (Δ12) C24,E26→S24, D26 (Δ24, 26) Rb Binding Consensus Domain (LXCXE) S31, S32→G31,G32 (Δ31, 32) Casein Kinase II Domain C91→W91 (Δ91) Putativemetal-binding motif

[0113] The PCR primers set out below were used to introduce the changes.

[0114] M12→L12 (Δ12)

[0115] Sense SEQ ID NO: 22

[0116] TACATTGCATGAATATTTGTTAGATTTGCAACCAG

[0117] Antisense SEQ ID NO: 23

[0118] CTGGTTGCAAATCTAACAAATATTCATGCAATGTA

[0119] C24,E26→S24,D26 (Δ24, 26)

[0120] Sense SEQ ID NO: 24

[0121] GAGACAACTGATCTCTACTCTTATGTCAATTAAATGACAGC

[0122] Antisense SEQ ID NO: 25

[0123] GCTGTCATTTAATTGATCATAAGAGTAGAGATCAGTTGTCTC

[0124] S31,S32→G31,G32 (Δ31,32)

[0125] Sense SEQ ID NO: 26

[0126] GAGCAATTAAATGACGGCGGAGAGGAGGAGGATGAA

[0127] Antisense SEQ ID NO: 28

[0128] TTCATCCTCCTCCTCTCCGCCGTCATTTAATTGCTC

[0129] C91→W9l (Δ91)

[0130] Sense SEQ ID NO: 27

[0131] ACACTAGGAATTGTGGGCCCCATCTGTTCTCAG

[0132] Antisense SEQ ID NO: 29

[0133] CTGAGAACAGATGGGGCCCACAATTCCTAGTGT

[0134] Multiple mutations within the E7 coding sequence were generatedby a series of site-directed mutagenesis reactions. At each step, themutagenesis was confirmed by sequence analysis. The isolated mutantproteins were examined for relative ability to activate CDK2 underconditions described above. Results are set out in Table 1 below. TABLE1 Amino Acid Changes versus CDK2 Activation % Control Activation AminoAcid Changes Exp. 1 Exp. 2 Δ12 96.7 Δ24, 26 86.7 81.3 Δ31, 32 77.1 Δ9181.7 78.7 Δ12; Δ24, 26 85.2 93.5 Δ24, 26; Δ31, 32 88.2 95.6 Δ24, 26; Δ9169.4 86.4 Δ12; Δ24, 26; Δ31, 32 83.1 Δ24, 26; Δ31, 32; Δ91 70.6

[0135] Results indicated that E7 can tolerate multiple alterations inamino acid sequence within previously determined functional domains andstill activate CDK2 kinase activity. This conclusion is demonstrated byE7 proteins having up to five mutated residues showing little change inthe ability to activate CDK2.

[0136] E7 Truncation Mutants

[0137] In an attempt to more fully elucidate E7 regions required forCDK2 activation, E7 fragments were tested in the above assay. HPV16-E7truncations were expressed using DNA wherein stop codons were introducedafter residues 87, 69, 48, 38, 27, and 15 in the full length coding E7sequence. The GST-HPV16 E7 pGEX-4T-3 expression construct was modifiedusing site-directed mutagenesis with a QuickChange™ kit (Stratagene) andthe proper sequence alterations were confirmed by sequence using aPerkin-Elmer ABI-PRISM™ . The truncated E7-GST fusion proteins werepurified and tested as described above. Primers used for to introducestop codons in the E7 coding region are set out below.

[0138] GST-16E7(1-15)

[0139] Sense SEQ ID NO: 40

[0140] GCATGAATATATGTTAGATTTGTAACCAGAGACAACTGATCTC

[0141] Antisense SEQ ID NO: 41

[0142] GAGATCAGTTGTCTCTGGTTACAAATCTAACATATATTCATGC

[0143] GST-16E7(1 -27)

[0144] Sense SEQ ID NO: 30

[0145] CTACTGTTATGAGCAATAAAATGACAGCTCAGAG

[0146] Antisense SEQ ID NO: 31

[0147] CTCTGAGCTGTCATTTTATTGCTCATAACAGTAG

[0148] GST-16E7(1-38)

[0149] Sense SEQ ID NO: 42

[0150] CAGAGGAGGAGGATGAAATATAAGGTCCAGCTGGACAAGCAG

[0151] Antisense SEQ ID NO: 43

[0152] CTGCTTGTCCAGCTGGACCTTATATTTCATCCTCCTCCTCTG

[0153] GST-16E7(1-48)

[0154] Sense SEQ ID NO: 32

[0155] GACAAGCAGAACCGGACTGAGCCCATTACAATATTG

[0156] Antisense SEQ ID NO: 33

[0157] CAATATTGTAATGGGCTCAGTCCGGTTCTGCTTGTC

[0158] GST-16E7(1-69)

[0159] Sense SEQ ID NO: 34

[0160] GCTTCGGTTGTGCGTATAAAGCACACACGTAGAC

[0161] Antisense SEQ ID NO: 35

[0162] GTCTACGTGTGTGCTTTATACGCACAACCGAAGC

[0163] GST-16E7(1 -87)

[0164] Sense SEQ ID NO: 36

[0165] GTTAATGGGCACACTATGAATTGTGTGCCCCATC

[0166] Antisense SEQ ID NO: 37

[0167] GATGGGGCACACAATTCATAGTGTGCCCATTAAC

[0168] Results indicated that deletion of HPV16 E7 residues between 48and 98 from the carboxy terminus caused a slight decrease in CDK2activation capability. HPV E7(1-87), HPV E7(1-69), and HPV E7(1- 48) allshowed similar degrees of activation of CDK2, with relative kinaseactivity of 76.67%, 82.45%, and 73.25%, respectively, compared toactivity in the presence of full length E7. HPV E7(1-38) alsodemonstrated relative kinase activity of approximately 72%. Truncationof residues 28-48 resulted in a significant drop in CDK2 activation, to28.64% in one experiment and to 52% in a repeat experiment of that offull length E7, suggesting that the majority of the CDK2 activationfunction of E7 resides in the amino-terminal portion of the protein. HPVE7(1-15), however, exhibited only 15% relative kinase active, indicatingthat the majority of the CDK2 promoting activity arises from sequencesfound in both CR1 and CR2 E7 regions. These results were confirmed bytruncation of the HPV16 E7 amino-terminus using the following PCRstrategy.

[0169] The pGEX-4T-3 GST-HPV16 E7 expression vector was also used astemplate to prepare amino terminal E7 deletion mutants. Residues 1-39 ofHPV16 E7 were eliminated using (i) a reverse PCR primer that hybridizedto the antisense strand immediately 5′ of the E7 gene in the pGEX-4T-3vector, and (ii) a forward second primer which hybridized starting atcodon 39 (and downstream from codon 39) of the E7 gene. PCR conditionswere identical to those used for carboxy terminal site-directedmutagenesis. The reaction resulted in the entire vector and E7 genebeing amplified with the exception of that region of E7 encoding aminoacid residues 1-38. The primers used were as follows.

[0170] GST-16E7(39-98) primers

[0171] Sense GATGGTCCAGCTGGACAAGC SEQ ID NO: 38

[0172] Antisense CATGGATCCACGCGGAACCAG SEQ ID NO: 39

[0173] The resulting PCR products were digested with DpnI to eliminatetemplate DNA, extracted with phenol-chloroform, ethanol precipitated,and transformed into E. Coli strain JM 109. Isolated clones wereprepared and the proper construct, missing the 5′-region of the E7 geneencoding residues 1-38, was confirmed by restriction endonucleasedigestion and sequence analysis. Expression of the GST-HPV16-E7(39-98)polypeptide was induced and the truncated protein was isolated usingglutathione affinity chromatography. Proper size was determined usingSDS-PAGE. Similarly GST-E7 constructs were generated encoding HPV16amino acids 9-98 and 16-98.

[0174] GST-HPV16-E7(39-98) assayed for the ability to activatecyclinA/CDK2 showed 28.6% of the activation activity of full lengthGST-HPV 16 E7. In a repeat experiment, GST-HPV16-E7(39-98) showed 18%activation potential. GST-HPV16-E7(9-98), however, showed 64% activationof full length E7, and GST-HPV16-E7(16-98) showed 53% activation. Theseresults, together with earlier results using the carboxy-terminaltruncation mutants, indicates that smaller regions within E7,particularly within the amino-terminal portion of the protein, arecritical for activation of cyclin-dependent kinases.

[0175] In view of these results, additional constructs were preparedencoding only internal amino acid regions of E7 or deletion mutantswherein internal E7 sequences were removed. Construct GST-16E7 (16-38)was prepared by introducing a stop codon in the construct encodingGST-16E7(16-98), while constructs GST-16E7(39-98) and GST-E7 (20-48)were prepared using a QuikChange™ mutagenesis kit using PCR primers toexclude amino terminal sequence using pGEX16E7 and pGEX16E7(1-48) astemplate DNA. Constructs bearing internal E7 amino Δ9-13, 13-15, Δ34-37,Δ21-37, Δ31-37, or Δ16-37 were deleted using the QuikChange™ mutagenesiskit according to the manufacturer's suggested protocol and pGEX-16E7 DNAas template.

[0176] Results from kinase assays using constructs GST-16E7 (16-38),GST-16E7(39-98), and GST-E7 (20-48) showed these E7 residues were ableto induce kinase activity to levels 53%, 18%, and 33%, respectively, ofthat by full length E7. Inducing activity for the various internaldeletion mutants is shown in Table 2. TABLE 2 Amino Acid Changes versusCDK2 Activation Amino Acid Changes % Control Activation Δ9-13 90 Δ13-1576 Δ34-37 80 Δ9-13, ΔA34-37 72 Δ22-26 85 Δ31-37 80 Δ22-26, Δ31-37 72

[0177] Combined, these result indicate that inactivation of theRb-binding domain, the casein kinase II domain or the zinc-bindingdomains results in only a small decrease in the ability of E7 toactivate CDK2 kinase activity. Similarly, deletion of both theRb-binding and casein kinase II domains resulted in a loss of only 28%of that determined for full length E7, and none of the internaldeletions from the CR1 and CR2 regions, alone or in combination,significantly affected activation by E7.

[0178] The E7 CR1 and CR2 regions show high degrees of similarity tonon-contiguous portions of adenovirus E1A protein and SV40 T antigen. Inview of this degree of similarity, it is expected that activation ofCDK2 is also a biological property of these and other viral proteins.

Example 6 E7 Interaction with CyclinA

[0179] In order to characterize E7 binding to the CDK2/cyclin complex,binding assays were carried out using CDK2 and cyclinA individually.

[0180] HighFive™ cells (Invitrogen) were infected with baculovirusexpressing GST-cyclinA (described above) or hemagglutinin (HA)-taggedCDK2 [Desai, et al., Mol. Cell. Biol. 3:571-582 (1992)].GST-cyclinA-expressing cells were lysed (4×10⁷ cells/ml) in lysis buffer(20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 100 mM NaCl, 5 mM NaF, 1 mM Na₃VO₄,0.5% NP-40, 10 μg/ml leupeptin, 4 μg/ml aprotinin, and 1 mM PMSF) andinsoluble material removed with centrifugation. GST-cyclinA expressionwas verified by SDS-PAGE and immmunoblotting. HA-CDK2-expressing cellswere lysed in low salt, hypotonic lysis buffer [10 mM HEPES, pH 7.4, 10mM NaCl, 1 mM EDTA, 0.1% NP-40, 1 mM DTT, 20 μM4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) and 1× Complete™proteinase inhibitor cocktail (Boehringer Mannheim)].

[0181] Glutathione agarose beads (25 μl) (Sigma) were preincubated with4% bovine serum albumin (BSA) in PBS, washed 3× in 1 ml lysis buffer,added to 1 ml GST-cyclinA lysate, and incubated at 4° C. for 1 hr withmixing. The beads were washed 4× with 1 ml lysis buffer, 2× with 1 mlbinding buffer (20 mM HEPES, pH 7.4, 10 mM KCl, 100 mM NaCl, 1 mM EDTA,1 mM EGTA, 0.1% NP-40, 12.5% glycerol, 1 mM DTT, and 1× Complete™proteinase inhibitor cocktail) containing 100 mM NaCl, and resuspendedin 0.4 ml binding buffer. Purified HPV16E7 (4 μg) was added to the beadsand incubation was carried out for 1.5 hr at 4 ° C. with agitation. Thebeads were washed 5× with 1 ml binding buffer, boiled in 2× Laemmlibuffer, and analyzed using SDS-PAGE and immunoblotting.

[0182] Crude HA-CDK2 lysate (0.2 ml) was diluted 1:5 in hypotonic lysisbuffer containing 150 mM NaCl and pre-cleared with 1 μg mouse IgG and 25μl Protein A/Protein G PLUS-Agarose (Santa Cruz Biotechnology, Inc.) for1 hr at 4 ° C. Immunoprecipitation of HA-CDK2 was carried out with 1 μganti-HA monoclonal antibody 12CA2 (Roche Molecular Biochemicals) and 20μl Protein A/Protein G PLUS-Agarose for 1 hr at 4 ° C. The beads werewashed 3× with 1 ml hypotonic lysis buffer containing 150 mM NaCl, 2×with 1 ml binding buffer, and resuspended in 0.4 ml binding buffer.Purified HPV16E7 (4 μg) was added to the beads and incubation wascarried out at 4° C. for 1.5 hr. The beads were washed in 5× with 1 mlbinding buffer, blotted in 2× Laemmli sample buffer, and analyzed bySDS-PAGE and immunoblotting.

[0183] Results showed E7 binding to beads containing GST-cyclinA, but nobinding to beads with immobilized HA-CDK2. These results suggest thatthe action of E7 on CDK2 enhanced kinase activity may be effectedthrough binding interactions with the cyclin component in theCDK2/cyclin complex. This binding interaction is consistent with thetheory that E7 is able to alter CDK2 substrate specificity in view ofthe fact that cyclins play a role in mediating CDK2 substratespecificity [Roberts, Cell 98:129-132 (1999)]. In one mode of action, E7binding may redirect CDK2 to viral substrates which participate in viralDNA replication. Alternatively, E7 binding can alter CDK2 specificityfor cellular substrates which either participate in promoting the onsetof host cell S phase or redirect cellular substrates to participate inreplicating viral DNA.

[0184] Numerous modifications and variations in the invention as setforth in the above illustrative examples are expected to occur to thoseskilled in the art. Consequently only such limitations as appear in theappended claims should be placed on the invention.

1 43 1 98 PRT Papillomavirus sylvilagi 1 Met His Gly Asp Thr Pro Thr LeuHis Glu Tyr Met Leu Asp Leu Gln 1 5 10 15 Pro Glu Thr Thr Asp Leu TyrCys Tyr Glu Gln Leu Asn Asp Ser Ser 20 25 30 Glu Glu Glu Asp Glu Ile AspGly Pro Ala Gly Gln Ala Glu Pro Asp 35 40 45 Arg Ala His Tyr Asn Ile ValThr Phe Cys Cys Lys Cys Asp Ser Thr 50 55 60 Leu Arg Leu Cys Val Gln SerThr His Val Asp Ile Arg Thr Leu Glu 65 70 75 80 Asp Leu Leu Met Gly ThrLeu Gly Ile Val Cys Pro Ile Cys Ser Gln 85 90 95 Lys Pro 2 28 DNAArtificial Sequence Description of Artificial Sequence primer 2cgggatccat gcgtggagaa acacctac 28 3 27 DNA Artificial SequenceDescription of Artificial Sequence primer 3 cgggatcctt acagtctagtagaacag 27 4 26 DNA Artificial Sequence Description of ArtificialSequence primer 4 cgggatccat gcatggaaga catgtt 26 5 26 DNA ArtificialSequence Description of Artificial Sequence primer 5 ccgctcgagttaggtcttcg gtgcgc 26 6 28 DNA Artificial Sequence Description ofArtificial Sequence primer 6 cgggatccat gcatggagat acacctac 28 7 31 DNAArtificial Sequence Description of Artificial Sequence primer 7ccgctcgagt tatggtttct gagaacagat g 31 8 30 DNA Artificial SequenceDescription of Artificial Sequence primer 8 gcctcgagat gtcagaaccggctggggatg 30 9 30 DNA Artificial Sequence Description of ArtificialSequence primer 9 gcggatcctt agaaggtaga gcttgggcag 30 10 27 DNAArtificial Sequence Description of Artificial Sequence primer 10gcctcgagct gcccaagctc taccttc 27 11 26 DNA Artificial SequenceDescription of Artificial Sequence primer 11 cggatcctat gtcaaacgtgcgagtg 26 12 27 DNA Artificial Sequence Description of ArtificialSequence primer 12 gcggatcctt agggcttcct cttggag 27 13 26 DNA ArtificialSequence Description of Artificial Sequence primer 13 aggatccttacgtttgacgt cttctg 26 14 28 DNA Artificial Sequence Description ofArtificial Sequence primer 14 gcctcgagat gtcaaacgtg cgagtgtc 28 15 30DNA Artificial Sequence Description of Artificial Sequence primer 15gcggatcctt acaccttgca ggcacctttg 30 16 27 DNA Artificial SequenceDescription of Artificial Sequence primer 16 gcctcgagaa aggtgcctgcaaggtgc 27 17 26 DNA Artificial Sequence Description of ArtificialSequence primer 17 gcggatcctt acgtttgacg tctctg 26 18 30 DNA ArtificialSequence Description of Artificial Sequence primer 18 gatcagatctcatgaaggag gacggcggcg 30 19 30 DNA Artificial Sequence Description ofArtificial Sequence primer 19 gcagatcttc agtggtggtg gtggtggtgg 30 20 31DNA Artificial Sequence Description of Artificial Sequence primer 20gggcggccgc atgtccccta tactaggtta t 31 21 27 DNA Artificial SequenceDescription of Artificial Sequence primer 21 gggcggccgc gtcagtcagtcacgatg 27 22 35 DNA Artificial Sequence Description of ArtificialSequence primer 22 tacattgcat gaatatttgt tagatttgca accag 35 23 35 DNAArtificial Sequence Description of Artificial Sequence primer 23ctggttgcaa atctaacaaa tattcatgca atgta 35 24 41 DNA Artificial SequenceDescription of Artificial Sequence primer 24 gagacaactg atctctactcttatgtcaat taaatgacag c 41 25 42 DNA Artificial Sequence Description ofArtificial Sequence primer 25 gctgtcattt aattgatcat aagagtagagatcagttgtc tc 42 26 36 DNA Artificial Sequence Description of ArtificialSequence primer 26 gagcaattaa atgacggcgg agaggaggag gatgaa 36 27 33 DNAArtificial Sequence Description of Artificial Sequence primer 27acactaggaa ttgtgggccc catctgttct cag 33 28 36 DNA Artificial SequenceDescription of Artificial Sequence primer 28 ttcatcctcc tcctctccgccgtcatttaa ttgctc 36 29 33 DNA Artificial Sequence Description ofArtificial Sequence primer 29 ctgagaacag atggggccca caattcctag tgt 33 3034 DNA Artificial Sequence Description of Artificial Sequence primer 30ctactgttat gagcaataaa atgacagctc agag 34 31 34 DNA Artificial SequenceDescription of Artificial Sequence primer 31 ctctgagctg tcattttattgctcataaca gtag 34 32 36 DNA Artificial Sequence Description ofArtificial Sequence primer 32 gacaagcaga accggactga gcccattaca atattg 3633 36 DNA Artificial Sequence Description of Artificial Sequence primer33 caatattgta atgggctcag tccggttctg cttgtc 36 34 34 DNA ArtificialSequence Description of Artificial Sequence primer 34 gcttcggttgtgcgtataaa gcacacacgt agac 34 35 34 DNA Artificial Sequence Descriptionof Artificial Sequence primer 35 gtctacgtgt gtgctttata cgcacaaccg aagc34 36 34 DNA Artificial Sequence Description of Artificial Sequenceprimer 36 gttaatgggc acactatgaa ttgtgtgccc catc 34 37 34 DNA ArtificialSequence Description of Artificial Sequence primer 37 gatggggcacacaattcata gtgtgcccat taac 34 38 20 DNA Artificial Sequence Descriptionof Artificial Sequence primer 38 gatggtccag ctggacaagc 20 39 21 DNAArtificial Sequence Description of Artificial Sequence primer 39catggatcca cgcggaacca g 21 40 43 DNA Artificial Sequence Description ofArtificial Sequence primer 40 gcatgaatat atgttagatt tgtaaccagagacaactgat ctc 43 41 43 DNA Artificial Sequence Description ofArtificial Sequence primer 41 gagatcagtt gtctctggtt acaaatctaacatatattca tgc 43 42 42 DNA Artificial Sequence Description ofArtificial Sequence primer 42 cagaggagga ggatgaaata taaggtccagctggacaagc ag 42 43 42 DNA Artificial Sequence Description of ArtificialSequence primer 43 ctgcttgtcc agctggacct tatatttcat cctcctcctc tg 42

What is claimed is:
 1. A method for identifying an inhibitor ofE7-induced CDK2 kinase activity, comprising the steps of a) measuringCDK2 kinase activity on a CDK2 substrate in the presence of humanpapillomavirus (HPV) E7, or a fragment thereof, and in the presence andabsence of a test compound, and b) identifying the test compound as aninhibitor of E7-induced CDK2 kinase activity when decreasedphosphorylation of the CDK2 substrate is detected in the presence of thetest compound compared to phosphorylation of the CDK2 substrate detectedin the absence of the test compound.
 2. The method according to claim Iwherein the HPV E7 fragment is selected from the group consisting ofamino acid residues 1 to 27, amino acid residues 1 to 38, amino acidresidues 1 to 48, amino acid residues 1 to 69, and amino acid residues 1to 87 of SEQ ID NO:
 1. 3. A method for identifying an inhibitor ofE7-induced CDK2 kinase activity comprising the steps of a) measuringCDK2 kinase phosphorylation of a CDK2 substrate; b) measuring increasedCDK2 kinase phosphorylation of the CDK2 substrate in the presence ofhuman papillomavirus (HPV) E7, or a fragment thereof, to determineE7-induced CDK2 kinase activity; c) measuring CDK2 kinasephosphorylation of the CDK2 substrate in the presence of HPV E7, or afragment thereof, and in the presence of a test inhibitor compound; andd) identifying the test compound as an inhibitor of E7-induced CDK2kinase activity when the increased phosphorylation measured in step (b)is reduced in the presence of the test compound.
 4. The method accordingto claim 3 wherein the HPV E7 fragment is selected from the groupconsisting of amino acid residues 1 to 27, amino acid residues 1 to 38,amino acid residues 1 to 48, amino acid residues 1 to 69, and amino acidresidues 1 to 87 as set out in SEQ ID NO:
 1. 5. The method according toone of claims 1, 2, 3, or 4 wherein the CDK2 substrate is selected fromthe group consisting of histone H1, HPV protein E1 and HPV protein E2.6. The method of claim 5 wherein measuring of CDK2 kinase activity iscarried out in the presence of a cyclin.
 7. The method of claim 5wherein the cyclin is selected from the group consising of cyclinA andcyclinE.
 8. A method for identifying an anti-viral agent comprising thesteps of a) identifying an inhibitor of E7-induced increase in CDK2kinase activity; b) measuring viral proliferation in the presence andabsence of the inhibitor identified in (a); and c) identifying theinhibitor as an antiviral agent when decreased viral proliferation isdetected in the presence of the inhibitor compared to viralproliferation in the absence of the inhibitor.
 9. A method for reducinghuman papillomavirus (HPV) E7-induced CDK2 kinase activity comprisingthe step of contacting an HPV infected cell with an inhibitor ofE7-induced CDK2 phosphorylation.
 10. A method for reducing humanpapillomavirus (HPV) E7-induced CDK2 kinase activity comprising the stepof contacting an HPV infected cell with an inhibitor of E7 binding toCDK2 kinase complex.
 11. A method for ameliorating human papillomavirus(HPV) proliferation comprising the step of administering to anindividual in need thereof an effective amount of an inhibitor of HPVE7-induced CDK2 kinase activity.
 12. A method for ameliorating humanpapillomavirus (HPV) proliferation coprising the step of administeringto an individual in need thereof an effective amount of an inhibitor ofHPV E7 binding to CDK2 kinase complex.
 13. The method of any of claims10 and 12 wherein binding of E7 to CDK2 kinase complex is effectedthrough interaction with a cyclin component of the activity.